Peter M. Smith

Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate

Submaximal stimulation with agonists generating inositol 1,4,5-trisphosphate (IP3) evokes cytosolic Ca2+ oscillations in many different cell types. In general, each Ca2+ rise is initiated from a specific region near the plasma membrane and then spreads as a wave throughout the cell. We now demonstrate that low (physiological) agonist concentrations evoke local cytosolic Ca2+ spikes in the secretory pole of single mouse pancreatic acinar cells that are particularly sensitive to blockade by the IP3 receptor antagonist heparin. These spikes can occur alone or repetitively or can precede longer lasting Ca2+ signals that spread throughout the cell. Intracellular IP3 application mimics these agonist actions. The short-lasting local Ca2+ spikes provide an economical signaling mechanism and are of physiological significance since they activate Ca(2+)-dependent Cl- and cation currents important for control of fluid secretion.

Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techniques into practical therapy.

Study design:Review.Objectives:To highlight the value of investigating the effects of putative therapeutic interventions in clinical spinal cord injury (SCI) in domestic dogs.Setting:England, UK.Methods:Many experimental interventions in laboratory rodents have been shown to ameliorate the functional deficits caused by SCI; the challenge now is to determine whether they can be translated into useful clinical techniques. Important differences between clinical SCI in human patients and that in laboratory rodents are in the size of the spinal cord and heterogeneity of injury severity. A further key issue is whether the statistical difference in outcome in the laboratory will translate into a useful difference in clinical outcome. Here, we stress the value of investigating the effects of putative therapies in clinical SCI in domestic dogs. The causes of injury, ability to categorise the severity and methods available to measure outcome are very similar between canine and human patients. Furthermore, postmortem tissue more rapidly becomes available from dogs because of their short lifespan than from human patients.Results:The role that investigation of canine SCI might play is illustrated by our preliminary trials on intraspinal transplantation of olfactory glial cells for severe SCI.Conclusions:This canine translational model provides a means of ‘filtering’ putative treatments before human application.Sponsorship:Our work described here was supported by the International Spinal Research Trust.

Ca2+ oscillations in pancreatic acinar cells : spatiotemporal relationships and functional implications

The pancreatic acinar cells are of particular interest for the study of cytosolic Ca2+ signals, since they are morphologically polarized and generate agonist-specific Ca2+ oscillation patterns. Recent data obtained by combining digital video imaging of Fura-2 fluorescence with patch-clamp whole-cell current recording have provided new information on the spatiotemporal relationships of the cytosolic Ca2+ signals and the Ca(2+)-activated ionic currents. Low agonist concentrations evoke repetitive short-lasting local Ca2+ spikes in the secretory pole region that activate shortlasting current spikes. In the case of acetylcholine stimulation the spikes are confined to this region. When cholecystokinin is used the shortlasting local spikes precede longer Ca2+ transients that spread to the whole of the cell. Infusion of non-metabolizable inositol trisphosphate analogues can mimic these responses. The shortlasting local Ca2+ spikes are particularly sensitive to blockade by the inositol trisphosphate receptor antagonist heparin. These results show that the secretory pole region has a particularly high sensitivity to inositol trisphosphate probably due to clustering of high affinity receptors.

Catalytic, asymmetric cyanohydrin synthesis mediated by lanthanide(III) chloride pybox complexes

Abstract Complexes formed between lanthanide trichlorides and 2,6-bis( substituted -2-oxazolin-2-yl)pyridine (pybox) ligands are effective catalysts for the enantioselective addition of trimethylsilylcyanide (TMSCN) to a range of aldehydes.

Morphological and Functional Characteristics of Acinar Atrophy and Recovery in the Duct-ligated Parotid Gland of the Rat

Although acinar atrophy occurs frequently in salivary diseases, the relationship between structural changes and functional decrements is not well-established, and the potential for recovery of histological and functional integrity has not been wholly quantified. We aimed, therefore, to develop further our understanding of pathological acinar atrophy. Stensens duct was ligated for periods up to six weeks and, in separate experiments, was de-ligated after two weeks and allowed to recover for up to two weeks. Qualitative and quantitative histological analyses were carried out. Additionally, the ability of enzymatically dispersed cells from ligated and de-ligated glands to respond to neuro-hormonal stimuli was also measured. The results confirmed that totally obstructed glands undergo a rapid, progressive severe atrophy amounting to absolute losses of over 85% of acinar tissue by two weeks. Acinar shrinkage and cell losses through apoptosis accounted for the glandular atrophy. Remaining intralobular epithelia consisted of extremely atrophic acini and numerous duct-like structures with intermediate forms. Dispersed cells from atrophic glands exhibited agonist-induced release of chloride similar to normal. Together, these structural-functional results confirm the persistent viability of acinar-like cells in the obstructed gland and suggest that the duct-like structures are derived from surviving atrophic acini. De-ligated glands exhibited a near-normal recovery of structure by two weeks. Their enzymatically dispersed cells responded normally to agonist stimulation. The results support the view that pathological atrophy is largely similar to physiological atrophy, providing a mechanism for acinar cell survival under adverse conditions, with the possibility of eventual recovery.

Acetylcholine-evoked calcium mobilization and ion channel activation in human labial gland acinar cells from patients with primary Sjögren's syndrome

Recent evidence has indicated that the salivary gland dysfunction associated with Sjögrens syndrome (SjS) is not necessarily due to immune‐mediated destruction of acinar tissue. SjS sufferers may possess substantial reserves of acinar tissue but nevertheless be incapable of maintaining salivary flow rates in the normal range. We have investigated the ability of isolated labial gland acinar cells from SjS patients to fluid secrete by measuring agonist‐evoked changes in intracellular Ca2+ ([Ca2+]i) using fura‐2 microfluorimetry and activation of K+ and Cl− channels using the patch‐clamp whole cell technique. We can confirm that stimulation with a super‐maximal dose of acetylcholine (ACh) increased [Ca2+]i equally in both control acinar cells and those derived from SjS patients. However, at submaximal concentrations, the dose–response curve for ACh was shifted to the right by approximately one order of magnitude in acinar cells from SjS patients compared to control acinar cells. Patch‐clamp measurements consistent with the presence of Ca2+‐activated K+ and Cl− conductances were obtained from both control acinar cells and those obtained from SjS patients. Dose‐dependent activation of the ion channels by acetylcholine was also right‐shifted in acinar cells from SjS patients compared to control cells. Our data show that labial gland acinar cells from SjS patients were capable of responding to agonist stimulation by mobilizing [Ca2+]i and activating K+ and Cl− channels consistent with the requirements of fluid secretion. However, the persistent loss of sensitivity to ACh observed in from SjS patients may account for the lack of saliva production observed in these patients in vivo.

Necrotizing Cerebellitis and Cerebellar Atrophy Caused by Neospora caninum Infection: Magnetic Resonance Imaging and Clinicopathologic Findings in Seven Dogs

BACKGROUND Adult dogs with neosporosis can develop a variety of neurologic signs. No area of predilection within the nervous system so far has been identified in adult dogs. OBJECTIVES To document neosporosis as a cause of progressive cerebellar ataxia and cerebellar atrophy in dogs. ANIMALS Seven client-owned dogs. METHODS Retrospective, descriptive study. RESULTS Age at diagnosis ranged from 1 year 6 months to 9 years 11 months. Neuroanatomic localization indicated cerebellar and brainstem disease in 6 dogs and a central vestibular lesion in 1 dog. In all 7 dogs, there was moderate to marked bilaterally symmetrical cerebellar atrophy, with the atrophied cerebellum being surrounded by a region of T2-weighted hyperintense and T1-weighted hypointense signal. Cerebrospinal fluid (CSF) analysis in all but 1 dog showed mononuclear pleocytosis and high protein concentration. Polymerase chain reaction testing for Neospora caninum performed on the CSF was positive in 4/5 dogs tested and there was a high titer of serum antibodies to N. caninum (> or = 1 : 800) in all 6 dogs tested. Postmortem examination in 1 dog confirmed cerebellar atrophy and multifocal nonsuppurative encephalitis with areas of malacia and leptomeningitis. All of the remaining 6 dogs were treated with some combination of clindamycin, trimethoprim, sulfadiazine, and pyrimethamine. Two dogs were euthanized because of deterioration or relapse of neurologic signs, but treatment of the remaining 4 dogs resulted in improvement (3 dogs) or resolution (1 dog) of neurologic signs. CONCLUSIONS AND CLINICAL IMPORTANCE Neosporosis is an important cause of progressive cerebellar ataxia and cerebellar atrophy in adult dogs.

The TRACTISS Protocol: a randomised double blind placebo controlled clinical TRial of Anti-B-Cell Therapy In patients with primary Sjögren's Syndrome

BackgroundPrimary Sjögren’s Syndrome (PSS) mainly affects women (9:1 female:male ratio) and is one of the commonest autoimmune diseases with a prevalence of 0.1 – 0.6% of adult women. For patients with PSS there is currently no effective therapy that can alter the progression of the disease. The aim of the TRACTISS study is to establish whether in patients with PSS, treatment with rituximab improves clinical outcomes.Methods/designTRACTISS is a UK multi-centre, double-blind, randomised, controlled, parallel group trial of 110 patients with PSS. Patients will be randomised on a 1:1 basis to receive two courses of either rituximab or placebo infusion in addition to standard therapy, and will be followed up for up to 48 weeks. The primary objective is to assess the extent to which rituximab improves symptoms of fatigue and oral dryness. Secondary outcomes include ocular dryness, salivary flow rates, lacrimal flow, patient quality of life, measures of disease damage and disease activity, serological and peripheral blood biomarkers, and glandular histology and composition.DiscussionThe TRACTISS trial will provide direct evidence as to whether rituximab in patients with PSS leads to an improvement in patient symptoms and a reduction in disease damage and activity.Trial registrationUKCRN Portfolio ID:9809 ISRCTN65360827.

An improved Williamson etherification of hindered alcohols promoted by 15-crown-5 and sodium hydride

Abstract 15-crown-5 greatly facilitates Williamson ether synthesis when sodium hydride base is used in THF solvent. This mild yet versatile procedure has been employed in the synthesis of new homochiral polyether ligands and bis-allylic ethers which are inaccessible by conventional methods.

THE MORPHOLOGY AND MORPHOMETRY OF THE NORMAL HUMAN TIBIALIS ANTERIOR MUSCLE

The light microscopic appearance of the human tibialis anterior muscle is described based on conchotome biopsy specimens from seven healthy volunteers and 20 patients who presented with myalgia but who had no evidence of neuromuscular disease. The morphometric characteristics of these normal muscles are documented and the similarities and differences between the appearances of the tibialis anterior and other muscles discussed.